Structure of the L5 Lipopolysaccharide Core Oligosaccharides of Neisseria meningitidis*

Three different oligosaccharides were isolated by mild acid hydrolysis of the lipopolysaccharides, ob- tained from Neisseria meningitidis serotype 5, and their structures were elucidated by combined chemical and physical techniques. The use of 500-MHz ‘H NMR in both one-dimensional and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the purified oligosaccharides were also degraded by chem- ical and enzymatic procedures to smaller fragments. The largest of the three original oligosaccharides is a triantennary partially 0-acetylated decasaccharide in which the largest antenna terminates in a la&o-N-neotetraose unit.

The meningococcal LPS' has been implicated in the immune response to natural infection (l), and at least 11 serotypes (Ll-Lll) have been identified (2,3). There is no apparent correlation between meningococcal serogroup, designated by meningococci having a common capsular polysaccharide, and LPS serotype, except that the LlO and Lll serotypes are exclusively associated with serogroup A organisms (4). The LPS serotype epitopes are located in the glycose moieties of the LPS (5), the latter having been identified as low molecular weight oligosaccharides of the R-type (6,7). By injecting rabbits with protein conjugates of the above oligosaccharides it has also been demonstrated (5) that they contain bactericidal epitopes. Structural studies (7,8)  reactivity exhibited by meningococci (3,5). However, the above structural studies did not address the phenomenon of heterogeneity among the LPS oligosaccharides associated with individual meningococcal serotypes (9, lo), which is probably the basis of even further immunologic diversity. This heterogeneity is generated either by structurally similar oligosaccharides having phosphoethanolamine groups in differing locations (8) or by glycose deletions from the oligosaccharides. This latter phenomenon was hypothesized to explain the molecular size heterogeneity exhibited by the LPS of individual meningococcal serotypes when run in sodium dodecyl sulfate gels (11,12), and this hypothesis has since been confirmed by chromatographic procedures on the isolated oligosaccharides (8,13). The isolation and structural determination of three different sized but structurally related oligosaccharides from the meningococcal L5 serotype confirms the above hypothesis. EXPERIMENTAL PROCEDURES*

Isolation
of Core Otigosuccharides-The heterogeneous nature of the core oligosaccharides of the L5 determinant was confirmed when 1% acetic acid hydrolysis of the LPS and gel filtration chromatography of the hydrolysate on Bio-Gel P-4 yielded three distinct products with K., 0.44, 0.59, and 0.65, designated oligosaccharides 1,2, and 3 in order of decreasing size ( Fig. 1).
Structure of Oligosaccharides 1, 2, and 3-The structures of oligosaccharides 1, 2, and 3 are shown in Fig. 2. Sugar analysis of 1 indicated that it was composed of D-galactose, D-glucose, 2-acetamido-2-deoxy-D-glucose, L-glycero-D-manno-heptose, and 3-deoxy-D-manno-octulosonic acid (KDO) in the molar ratio of 2:3:2:2:1. In addition the 'H NMR spectrum of 1 indicated that it also contained 0-acetyl groups (6 = 2.19 ppm) in a molar ratio of -0.4. Following removal of these groups with sodium hydroxide, the 'H NMR of de-Oacetylated 1 (Table I) was in agreement with the sugar analysis, i.e. two signals at 6 2.041 and d 2.119 ppm were indicative of N-acetyl groups assigned to the two D-glucosamine residues b and i, respectively. Also, nine signals in the proton anomeric region indicated that 1 contained at least 9 sugar residues with 5 of them, at 6 (Table II) indicated that it contained terminal nonreducing D-galactose, D-glucose, and 2acetamido-2-deoxy-D-glucose residues, D-galactose linked at O-3, two D-glucose residues, and one 2-acetamido-2-deoxy-Dglucose residue linked at O-4. Both heptoses were branch points, one of them substituted at O-3 and O-4, the other one at O-2 and O-3. KDO was linked at O-5 as it is in the L3 determinant (7). In order to obtain information on the sequence, a number of chemical and enzymatic degradations were performed on 1. First, it was anticipated from the structural information obtained for the L3 core determinant and from the results of the methylation analysis of 1 that 1 would also contain the same lacto-N-neotetraose unit from which P-D-Galp-l-+4-P-D-Glcp-NAc-l-+3-D-Galp would be hydrolyzed by endo-@galactosidase of Escherichia freundii (23).
Indeed when 1 was treated with the enzyme, two products of hydrolysis could be isolated by gel filtration on Bio-Gel P4 and identified following methylation and 'H NMR analyses. The fastest moving (larger) component had K., = 0.65 identical to that of 3, and 'H NMR analysis of this compound indicated that indeed it did have a structure identical to that of 3 (Fig. 2). The slowest moving (smaller) component (6) was shown to be a reducing trisaccharide having the structure P-D-Galp-I-+4-P-D-GlcpNAc-l+3-D-Galp by methylation (Table   II) and 'H NMR analysis (Table I). As shown above, 3 is a product resulting from both the partial hydrolysis (1% acetic acid) of the native L5 LPS and the endo-p-galactosidase treatment of oligosaccharide 1.

Methylation analysis of 3 indicated that it contained all methylated
sugars present in 1 (Table II) except for those  corresponding to unit 6 and for the appearance of one terminal glucose unit and disappearance of the 0-4-linked glucose residue present in 1. These results were in agreement with the known specificity of endo+?-galactosidase from E. freundii which cleaves the /3-D-Galp-1+4-P-D-Glcp linkage present in various glycosphingolipids (23). The sequence and anomeric configurations of the individual residues of 3 (its 0-deacetylated and NaBH, reduced form) were ascertained from 'H NMR (NOE) data. The chemical shifts of some of the protons associated with the individual residues of modified 3 were assigned by two-dimensional (H,H) COSY using both one-step (14) and two-step relayed coherent transfer (15) and are listed in Table III. Following assignments, the individual anomeric signals of each of the residues (g, h, i, f, e, d) in 3 (Fig. 2) were selectively irradiated, and the observed NOE values are listed in Table IV.

Irradiation of H-l (d) gave enhancements on its own H-3 and H-5 protons as well as on H-4 (e) indicating that the terminal glucopyranosyl residue (d) is in the B-D-configuration and is linked to O-4 of the adjacent D-ghCOpyranOSyl residue (e). When H-l (e) was irradiated it gave enhancements on H-3 (e) and H-5 (e) consistent with e being in the @-D-configuration and on H-4 (f) indicating that e was linked to O-4 of the next L-glycero-D-manno-heptopyranosyl residue (f). Irradiation of H-l (f) gave enhancements on H-2 (f) indicating that f is in the a-D-manno configuration;
other enhancements were observed that could belong to the borohydride-reduced (open chain) form of the KDO residue (j), but we could not be certain of the assignments of these signals. When H-l (h) was irradiated, it gave enhancements on H-2 (h) consistent with h being in the a-D-configuration and on H-3 (g) indicating that the terminal a-D-glucopyranosyl residue h was linked to O-3 of its adjacent L-glycero-a-D-manno-heptopyranosyl residue (g). Now when H-l (g) was irradiated enhancements on both H-2 (g) and H-3 (f) were observed, indicating that g is in the a-D-mannO configuration and that it is linked to O-3 of the L-glycero-D-manno-heptopyranosyl branch point residue (f). Finally when H-l (i) was irradiated, enhancements on both H-2 (i) and H-2 (g) were observed consistent with the terminal 2-acetamido-2-deoxy-D-glucopyranosyl residue i being in the a-D-configuration and linked through O-2 of its adjacent L-glycero-D-manno-heptopyranosyl residue (g). The NOE data support the sequence of glycoses of 3 shown in Fig.  2. It is interesting to note that the structure of 3 is part of that of the L3 core determinant (7) except for an additional a-D-glucopyranosyl residue linked at O-3 of the heptopyra-nosy1 side chain residue (g) and also for an additional P-Dglucopyranosyl residue linking the lacto-N-neotetraose unit to the inner core of 1. The "native" oligosaccharide 3 as well as the resulting digestion product of 1 by endo-/3-galactosidase are both 0-acetylated (-30%) on an as yet undetermined position on the terminal 2-acetamido-2-deoxy-a-D-glucopyranosyl residue (i); this chemical evidence was originally obtained for 1, 2, and 3 by FAB-MS upon analysis of their (positive mode) mass spectra (24). The final structure of 1, shown in Fig. 2, is of course the result of linking the reducing trisaccharide 6 to heptasaccharide 3. To confirm this we performed deamination studies on N-deacetylated 1. Following treatment with sodium nitrite in acetic acid, the products of deamination of N-deacetylated 1 were purified on Bio-Gel P4, and two major products were obtained. The largest fragment identified as 4 (Fig. 2) was analyzed by methylation (Table II) and 'H NMR analyses (Table I). By comparison with the methylation analysis of 3, that of 4 contained one more additional terminal galactose residue, and in addition one of the original 2,3,4,6tetramethylglucose residues found originally in 3 was transformed into a 2,3,6-trimethylglucose indicating that the terminal galactopyranosyl residue (c) is linked to O-4 of the glucopyranosyl residue (d). Also, the 4,6,7-trimethylheptopyranosyl residue present in the methylation analysis of 3 was now replaced by a 2,4,6,7-tetramethylheptopyranosyl residue indicating that the terminal 2acetamido-2-deoxy-glucopyranosyl residue (i) was indeed linked to O-2 of residue g as previously indicated by 'H NMR (NOE) data on 3. The smallest deamination product identified as 5 was shown to have the structure depicted in Fig. 2 following methylation and 'H NMR analyses. Additional confirmatory evidence that terminal galactose (c) of 4 ( Fig. 2) was linked to O-4 of the glucopyranosyl residue (d) was obtained when 4 was treated with a P-Dgalactosidase. Indeed after treatment with the enzyme, 4 has its terminal fi-D-galactose residue (c) removed, and a new product identified as 7 was characterized following methylation and 'H NMR analyses. It had the structure depicted in Fig. 2, where the 0-4-linked glucose residue (d) in 4 had now become a terminal nonreducing end. Based on the structural results obtained from both treatment of 1 with endo-@-galactosidase and deamination studies, a single structure for 1 as depicted in Fig. 2 was proposed. The entire structure differs from that of the L3 core (7)  residue (h) linked to O-2 of the heptopyranosyl side chain residue (g). The oligosaccharide is partially 0-acetylated on residue (i) and does not contain phosphoryl substituents as in the case of the L3 core determinant (7). Finally the third product with K,, 0.59 identified as 2 obtained from the acetic acid hydrolysis of the L5 LPS is an oligosaccharide whose structure depicted in Fig. 2 is an intermediate between that of 1 and that of 3. It differs from that of 1 by lacking the terminal P-D-Gall+4P-D-GlcpNAc disaccharide unit. The compositional analysis as well as the sequence of residues in oligosaccharides 1, 2, and 3 was totally (for the composition) and in part (for the sequence) confirmed by FAB-MS studies (24).

DISCUSSION
The structures of the three oligosaccharides isolated from the L5 serotype LPS are shown in Fig. 2. The structure of the largest oligosaccharide has been previously reported (8) and has terminal lacto-N-neotetraose on its longest antenna. This structural feature has also been identified on the oligosaccharides obtained from the LPS of the L2 (8) and L3 (7) serotypes and the fact that the LPS from which they were obtained exhibit predominant serotype specificity (5) confirms that lacto-N-neotetraose, despite its large size, is not immunodominant. Thus the serotype epitopes of the meningococcal LPS reside in the heptose-containing inner core of its oligosaccharide moieties (8). The nonimmunogenicity of the lacto-l\rneotetraose unit is probably due to immune tolerance because it is a known human and animal tissue antigen (25, 26). The largest of the L5 oligosaccharides differs in structure from that of L3 (7) by having two additional D-glucopyranosyl residues linked to its inner core. A terminal ol-D-glucopyra-nosy1 residue is linked to O-3 of one L-a-D-heptopyranosyl residue, and an interchain @-D-ghCOpyranOSy1 residue is linked to O-4 of the other L-a-D-heptopyranosyl residue. The lacto-l\r-neotetraose unit is linked to O-4 at this latter P-Dglucopyranosyl residue. These structural features play a dominant role in both the serotype specificity of the L5 LPS and also account for cross-reactions with both the L2 and LlO serotype LPS (5).
The isolation of three different oligosaccharides from the LPS of the L5 serotype confirms the heterogeneity of these structures on LPS obtained from meningococci of the same serotype. This phenomenon had been hypothesized to explain the heterogeneity exhibited by the individual serotype LPS of both meningococci (9, 10) and gonococci (12) when subjected to sodium dodecyl sulfate gel chromatography (9,12), and confirmation of this hypothesis was later obtained by analytical and oligosaccharide chromatographic procedures (8,13). However, the structural basis of this heterogeneity was not previously established. The three oligosaccharides from the LPS of the L5 serotype are structurally identical except for glycose deletions. Interestingly these deletions occur exclusively from the longest antenna of the L5 oligosaccharide whereas glycose deletions from the inner heptose core would have produced oligosaccharides associated with LPS of different serotypes (8). Also, it is interesting to note that glycose deletions from the long antenna of the L5 oligosaccharide create new and probably more immunogenic epitopes due to the destruction of the lacto-N-tetraose unit. This is consistent with the fact that of all the meningococcal serotype LPS, only in the case of L5 serotype is the smallest component on SDS gels the major antigenic component (27).
All the three oligosaccharides obtained from the L5 serotype LPS are partially 0-acetylated on their terminal 2-acetamido-2-deoxy-cu-D-glucopyranosyl residues. This is the first report of the presence of 0-acetyl substituents in the neisserial LPS, the identification of which suggests a further mechanism by which meningococci modulate their surface glycose structures. The identification of 0-acetyl substituents also raises the question as to whether in the L5 LPS the oligosaccharides are completely 0-acetylated. Certainly it is reasonable to assume that 0-acetyl groups could be partially removed under the mild hydrolytic conditions used to obtain the oligosaccharides from the LPS.